Genomic instability diseases like Fanconi Anemia (FA) represent rare genetic models for human susceptibility to cancer. FA is a multi-gene disease characterized on the cellular level by chromosomal abnormalities and a hypersensitivity to replication-blocking DNA lesions, particularly DNA interstrand crosslinks (DNA ICLs). A central member of the FA pathway is the FANCD2 protein that is directly recruited to DNA ICLs and stalled replication forks. Accumulating evidence suggests that FANCD2 then recruits other FA and non-FA DNA repair factors that use homologous recombination (HR) repair mechanisms to mediate the removal of replication- blocking lesions such as DNA ICLs, and to promote replication fork recovery. Based on our new results, we predict a protein named EMSY to be a novel functional partner of FANCD2. Whether EMSY functions in any cellular DNA damage responses is not known. We discovered that EMSY is recruited to chromatin in a DNA damage- and strictly FANCD2-dependent manner. Strikingly, EMSY-deficient cells are as hypersensitive to DNA ICLs and to replication stress as FANCD2-deficient cells. Based on our preliminary results, we hypothesize that EMSY and FANCD2 form a novel protein complex that promotes HR- dependent DNA ICL repair and HR-mediated replication fork recovery. We will test this hypothesis as follows: Aim 1: Determine the role of EMSY/FANCD2 in HR-mediated DNA ICL repair. We will test if EMSY and FANCD2 act in concert to promote cellular DNA ICL resistance and to support molecular steps during the HR- repair of DNA ICL-associated DNA double strand breaks (DSBs). Aim 2: Determine the role of EMSY/FANCD2 in HR-mediated replication fork recovery. We will test if EMSY and FANCD2 act in concert to promote cellular resistance to aphidicolin-triggered replication fork stalling and to support molecular steps during HR factor-mediated protection and restart of replication forks. Additionally, we will test if six known FANCD2 patient mutations cause cellular deficiencies in replication fork recovery by interrupting FANCD2/EMSY complex formation. These approaches will elucidate how the currently uncharacterized EMSY protein function in the cellular DNA damage response and reveal a new layer of complexity within the FA pathway-associated DNA repair network. We expect our findings to significantly contribute to an understanding of the FA pathway's role in maintaining genomic stability and in protecting cells from mutagenic events. This in turn will impact research on cancer predisposition and chemotherapeutic treatment strategies.